Current Agriculture Research Journal Vol. 2(2), 83-88 (2014) Stability of Metarhizium acridum Based Biopesticide in Operational Conditions in Senegal PAPA MADIALLACKÉ DIEDHIOU1*, KEMO BADJI2, ROKHAYA FAYE3 and PAPA IBRA SAMB3 1Faculté d’Agronomie, Université Gaston Berger ; Saint-Louis - Sénégal. 2Plant Health and Quarantine Officer, Direction de la Protection des Végétaux, Dakar- Sénégal 3Département de Biologie Végétale, Université Cheikh Anta Diop de Dakar, Sénégal. http://dx.doi.org/10.12944/CARJ.2.2.03 (Received: November 17, 2014; Accepted: November 25, 2014) ABSTRACT Grasshoppers and locusts are important pests in the Sahel zone with a big economic and environmental impact. The move towards biological control methods represents an important option to mitigate environmental impact linked with the use of insecticides for their control. Metarhizium acridum, a locust pathogenic fungus that gave excellent test results in controlling grasshoppers, was further developed as biopesticide. The stability of the manufactured biopesticide in operational conditions was tested. In the present study, the germination of spores and mycelial growth of M. acridum were higher at 28 °C and 33 °C. Higher temperatures led to a significant decrease of these biological parameters. The sporulation of M. acridum is abundant at 28 °C and 33 °C but was completely inhibited at 37 °C. Furthermore, M. acridum based biopesticide showed a good infection rate for the dry spores formulation for 3 months of storage in all test sites. In the oil formulation in contrast, infectivity rate dropped quickly to a very low level after one month storage in the inland sites and remained good in Dakar. High temperatures and low moisture, away from the coast were speculated to be the reasons. Key words: Metarhizium acridum, Biopesticide, Grasshoppers, Locusts, Oil formulation,Dry spore formulation. INTRODUCTION in 2004, a total of 1,040 billion CFA was needed to mitigate environmental pollution (Locustox, 2009). Grasshoppers and locusts are major pests in most countries of the Sahel zone like Senegal. Gradually, environment friendly alternatives In invasion years, their control requires annually pesticides are being developed to control locusts. large amounts of synthetic pesticides sprayed over Biopesticides like those having insect pathogenic about 1,000,000 ha per year (Launois Luong et al., microorganisms as active agent are increasingly 1988). In locust invasion years, about 2,000,000 developed for operational use in several countries ha are treated with pesticides to achieve a control of the Sahel zone. over the large migrating locusts and grasshoppers populations (Anon., 1997). Besides negative In West Africa, particularly in Senegal, economic impact, should be added other impacts Metarhizium acridum, a locust pathogenic fungus on the environment and on health of humans as gave excellent test results in controlling grasshoppers. well as on animals. With respect to negative impact The fungal strain was further formulated to a on environment, the decontamination of 1 ton of biopesticide, found to be very effective against sand contaminated with pesticides is estimated at locusts and grasshoppers. Those insects belong to 26,000,000 CFA. With a total of 40,000 tons of soil the order Orthoptera, and consist among others, to be decontaminated in pesticide storages sites with Schistocerca gregaria, Oedaleus senegalensis, 84 DIEDHIOU et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014) Hieroglyphus daganensis, Locusta migratoria, etc. Study sites The spores of Metarhizium acridum represent the The study of the viability of the spores of active ingredient of the bioinsecticide, intended for Metarhizium acridum in different formulations of locust control. However, the widespread use of this the biopesticide during storage was carried out in biopesticide in operational conditions in Senegal is the storage facilities of “Direction de la Protection facing a technical problem due to a lack of stability des Végétaux” (DPV), the National Plant Protection of the active ingredient during storage. In fact, the Organization. Those storage facilities are located biological effectiveness of the product in operational in different cities scattered over the whole country, conditions drops below the critical rate of 85% like Richard Toll, Louga, Gossas, Nioro du Rip and infectivity (Jaronski, 2000) in different environments. Dakar. They are designed for almost permanent Nonetheless, the feasibility of this control method, in storage of insecticides for locust control targeting particular during the hot rainy season, characterized O. senegalensis and S. gregaria. The mean with rapid buildup of locust populations, and a temperatures and relative humidity recorded in the worsening of roads and transportation systems, storages facilities during the experiment are listed requires the pesticides to be stored in close proximity in Table 1. to the agrosystems at risk and applied as needed. Storage test The present work therefore was intended to Samples the biopesticide in dry spores look at the influence of temperature on the survival formulation as well as oil formulation were placed of the active ingredient of a Metarhizium acridum in test tubes. For dry spores formulation, 1 g of based biopesticide in different formulations, with powder was sampled and 5ml of oil formulation. In special focus on the storage facilities of the National each storage facility, 5 tubes for oil formulation and Plant Protection Organization over the country. 5 other tubes for dry spores formulation were kept. The germination rate was recorded before storage MATERIALS AND METHODS and every 30 days, as described by Moore et al., (1996). For the germination test, 1 ml was sampled The fungal biocontrol agent from oil formulation containing tubes and 0.1 g from Metarhizium acridum strain IMI 330189 is dry formulation treatment. The samples were kept the active ingredient of the biopesticide. It is a fungus in small vials closed with parafilm and placed in a of the class ascomycete with Metacordyceps sp humid chamber for 24 hours (Moore et al., 1996). as teleomorph. Species of the genus Metarhizium Thereafter, 10 ml sterile water and a droplet of live naturally in the soil litter almost everywhere in Tween 80 were added prior to centrifugation for 5 the world. Metarhizium acridum strain IMI 330189 minutes at 2500 rotations / min. The solvent was was isolated from a parasitized Ornithacris cavroisi gently removed and the pellet, composed with found in Niger (Kooyman, 2007). The biopesticide is spores is mixed with 10 ml sterile water and Tween formulated as a powder composed with dry spores or 80. This spore suspension is used for germination spores mixed with vegetable oil as oil formulation. test by plating a 20, 50 and 100 µL into petri dishes containing Sabouraud Dextrose Agar culture media. Table. 1: Mean temperatures and moisture in the storage facilities of DPV during the test period Site hosting pesticide Temperatures (°C) Relative humidity (%) storage facility Maxima Minima Mean Maxima Minima Mean Gossas 37.00 23.63 29.69 66.70 32.10 59.58 Dakar 34.01 23.63 30.22 70.70 34.80 60.64 Louga 33.17 23.24 28.87 30.4 27.4 26.14 Nioro 37.88 24.01 30.13 28.52 22.6 24.97 R. Toll 37.88 22.48 29.58 25.42 16.61 22.68 DIEDHIOU et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014) 85 For the oil formulation, the sampled volume was spores extracted from the biopesticide. The number completed to 10 ml with sterile water and Tween 80. of germinated conidia was determined by over a 20 The mixture was centrifuged and the pellet collected conidia sample, repeated 10 times after 6 hours, 12 and suspended in 10 ml sterile water and Tween 80 hours, 18 hours and 24 hours of incubation. before being plated onto the culture media. Data analysis Effect of temperature on spore germination, An analysis of variance was performed mycelial growth and sporulation for data with a normal distribution. Data expressed For the study of mycelial growth and in percentages were first normalized by arcsinus sporulation ability, petri dishes with the culture media transformation. The statistical analysis software were inoculated with a 6 mm diameter of 4-5 days Minitab 13th edition for Windows was used. Means old culture of the fungus and incubated at different were separated using the Student test & Newman temperatures. For sporulation performance mature Keuls at 5 %. conidia were harvested after 3 weeks of incubation at constant temperatures in the dark. To collect the RESULTS spores, a fine brush and 10 ml sterile water and Tween 80 were used to prepare a suspension of Effect of different temperatures on germination spores. The concentration of the suspension of of spore of M. acridum spores obtained was determined using the grid cell The spore germination of M. acridum at of Neubauer (Tefera and Pringle, 2003). different temperatures gave variable results (Fig. 1). The highest rates were obtained at 33 °C and 28 °C Mycelial growth of the fungus at different with 18% and 21.12% respectively after only 6 hours temperatures was measured by taking the average of incubation. The germination rate reached over of two perpendicular diameters of colony in the petri 90% after 24h. When temperatures reached 35 °C dish. The diameter was measured every 24 hours and 37 °C, the germination rate did not cross the 10% for 11 days (Tefera and Pringle, 2003). bar after 24 hours. The analysis of variance showed a highly significant difference between treatments (p The effect of a range of constant H” 0.000). temperatures was evaluated on the germination of Fig. 1: Influence of different temperatures on spore germination of M. acridum (n = 10; p< 0.000) 86 DIEDHIOU et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014) Effect of different temperatures on mycelial highly significant differences between treatments (p growth of M.